All of the publications, patents and patent applications cited within this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
There is an increasing demand for a small scale array-based and/or microfluidic device that processes micro- or nano-volumes of sample, with time and cost savings arising from miniaturization. Prior art approaches to miniaturised polymerized chain reactions (“PCR”) make use of open or enclosed chambers or flow through zones/channel networks with appropriate temperature regulation; some have on-board silicone rubber-based or magnetic-based valving and/or pumping. Although potentially powerful approaches, they have challenges such as pressure sealing, prevention of evaporation, pressure buffering, chemical interference through surface interactions, or contamination via the porous and gas permeable membranes used in pumps and valves.
Performing PCR in a colloidal hydrogel matrix (hereafter termed “hydrogel”) confers a multitude of advantages. For example, the DNA, polymerase enzyme and other PCR reagents have reduced access to the device materials' surfaces where they may be adsorbed, absorbed, poisoned or otherwise rendered inactive; and are kept within close proximity to each other without the need for valves or pumps. Further, any contaminant solutes from device materials have reduced access to the PCR reaction.
As first introduced by Chetverin et al., gels provide a productive medium for PCR: see for example U.S. Pat. No. 5,616,478; which is herein incorporated by reference in its entirety. As described therein, PCR was confined to circular spots in a hydrogel sheet where the initial DNA or RNA templates, formed “molecular polonies” (short for polymerase colonies and named for their similarity to the growth of bacterial colonies in agar). As the initial amount of DNA can be accurately estimated by counting the number of polonies, Mitra et al. (Mitra, R. D. et al; Nucleic Acid Research 1999, 27, e34) performed DNA amplification in a thin acrylamide film polymerized with all the reagents along with plasmid. DNA as their template. In an alternate approach, Strizhkov et al. (Strizhkov, B. N. Et al; Biotechniques 2000, 29, 844-857) used nanoliter hydrogel pads to immobilize primers for PCR. Single Nucleotide Polymorphisms (SNPs) in cDNA were detected with polony technology by Butz et al. (Strizhkov, B. N. et al; 2000, 29, 844-857)
Recent advances have demonstrated that PCR can be performed in small volumes normally associated with microfluidic (sub-100 μl) reactions without the use of immobilized primers, as well as demonstrating that nucleic acids of sub-50 base pairs to megabase size can be administered to the surface of the hydrogel matrix with the nucleic acids capable of acting as a template to the remaining PCR reaction components internal to the hydrogel. See for example WO2012003579 by Atrazhev et al, or WO2012027832 by Atrazhev; which are herein incorporated by reference in their entirety.
The present art is in need of a means to perform analysis of amplicons generated by PCR, such as melting curve analysis (“MCA”) without imposing additional steps for the transfer or handling of PCR product and to do so with a simplified process for administration of fluids for interrogation through PCR constrained within the hydrogel.